JP7462411B2 - A method for producing a three-dimensional gingival epithelium model induced in a periodontal disease-like state - Google Patents
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Description
本発明は、三次元歯肉上皮モデルの組織内に歯周病原性細菌を感染させた歯周病様状態に誘導された三次元歯肉上皮モデルの製造方法に関する。 The present invention relates to a method for producing a three-dimensional gingival epithelium model induced into a periodontal disease-like state by infecting the tissue of the three-dimensional gingival epithelium model with periodontal pathogenic bacteria.
歯周病は、生活習慣病の一つで、口腔内の細菌の感染によって引き起こされる炎症性疾患である。口腔内の常在細菌３００～７００種類が、歯周病に関連が深い順にクラス分けがなされており、最も関連が深いとされる３菌種（ポリフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ）、トレポネーマ・デンティコーラ（Ｔｒｅｐｏｎｅｍａ ｄｅｎｔｉｃｏｌａ）、タンネレラ・フォーサイシア（Ｔａｎｎｅｒｅｌｌａ ｆｏｒｓｙｔｈｉａ）は、「レッド・コンプレックス」と呼ばれ、これらの菌にアグリゲイティバクター・アクチノミセテムコミタンス（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ））を含めて、歯周病の発病に関連の深い菌種とされている。 Periodontal disease is a lifestyle-related disease, an inflammatory condition caused by bacterial infection in the oral cavity. The 300 to 700 types of bacteria that normally inhabit the oral cavity are classified in order of their relevance to periodontal disease, and the three most closely related species (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) are known as the "red complex," and these bacteria, along with Aggregatibacter actinomycetemcomitans, are considered to be closely related to the onset of periodontal disease.
近年、全身疾患と歯周病の関連性が指摘されており、呼吸器系疾患、心疾患、糖尿病などとの関連が報告されている。歯茎から侵入した歯周病原性細菌は血管を介して全身に影響を与える可能性が示唆されており、口腔内における歯周病原性細菌の侵入を食い止めることが重要であると考えられている。 In recent years, the relationship between systemic diseases and periodontal disease has been pointed out, and links with respiratory diseases, heart diseases, diabetes, etc. have been reported. It has been suggested that periodontal pathogenic bacteria that invade through the gums may affect the entire body via the blood vessels, and it is considered important to stop the invasion of periodontal pathogenic bacteria in the oral cavity.
このようなことから、歯周病原性細菌の増殖及び組織内への侵入を防ぎ、歯周病を予防する事が生活習慣病を予防することに繋がると考えられている。
歯周病の予防、治療法を評価するにあたり、歯周病原性細菌に対する抗菌試験や、歯肉由来細胞を用いた試験法に関する研究が進められている。しかしながら、これら試験では、歯周病原性細菌に対する独立した抗菌試験や、歯肉由来細胞の応答を評価する試験が一般的であり、歯周病原性細菌と歯周組織をｉｎ ｖｉｔｒｏ系で組み合わせて試験している例は少ない。
For these reasons, it is believed that preventing the proliferation and invasion of periodontal pathogenic bacteria into tissues and preventing periodontal disease will lead to the prevention of lifestyle-related diseases.
In order to evaluate the prevention and treatment of periodontal disease, research is being conducted on antibacterial tests against periodontal pathogenic bacteria and test methods using gingival cells. However, these tests generally involve independent antibacterial tests against periodontal pathogenic bacteria or tests evaluating the response of gingival cells, and there are few examples of tests combining periodontal pathogenic bacteria and periodontal tissues in an in vitro system.
近年、動物実験に代わる実験方法の１つとして、三次元に構築された人工組織モデルを用いたｉｎ ｖｉｔｒｏ試験が普及しており、様々なタイプの人工組織モデルの作製およびこれらを用いた試験方法について研究されている。
オーラルケア分野では、動物実験が一般的であった背景もあり、歯肉上皮細胞を立体構築した三次元組織モデルについては十分に研究されていない。三次元組織モデルは個体差が少ないこともあり、抗菌剤を含む製剤および薬剤評価試験への活用が期待できる。さらに動物愛護の観点からも、今後は三次元組織モデルの活用が望まれている。
三次元歯肉モデルに歯周病菌を接種し、歯周病原性細菌を歯肉組織内に侵入させる状態を作り出すことができれば、これまでの独立した菌または細胞の試験のレベルからより生体環境に近い状態を模すことができる。
In recent years, in vitro testing using three-dimensionally constructed artificial tissue models has become widespread as an alternative experimental method to animal testing, and research is being conducted on the creation of various types of artificial tissue models and testing methods using these.
In the field of oral care, animal testing has been common, and there has been insufficient research into three-dimensional tissue models that construct gingival epithelial cells in three dimensions. As three-dimensional tissue models have little individual variation, they are expected to be used in formulations and drug evaluation tests, including antibacterial agents. Furthermore, from the perspective of animal welfare, the use of three-dimensional tissue models is expected in the future.
If we could inoculate periodontal bacteria into a three-dimensional gingival model and create conditions that would allow periodontal pathogenic bacteria to invade gingival tissue, we could mimic conditions that are closer to the in vivo environment than previous tests of independent bacteria or cells.
類似した例としては、三次元口腔粘膜モデルへ真菌である口腔カンジダ菌を感染させた報告がある（非特許文献１）。
しかしながら、歯周病原性細菌自体を歯肉組織に感染させることにより歯周病様状態を誘導する方法については報告がない。
対象とする歯周病原性細菌は、一般的に嫌気性条件を好む菌が多く、三次元歯肉モデルの培養条件下では生育しにくいことが考えられる。また、口腔粘膜モデルと異なり、歯肉上皮モデルは角化した層、すなわち、角化組織を有していることから容易に歯周病原性細菌を進入させることは困難であると考えられた。
そこで、三次元歯肉上皮モデルを用いて、歯周病原性細菌を感染させた歯周病様状態のモデルを作製する方法を考案した。歯周病原性細菌が感染した歯周病様状態により近いモデルを作製することができれば、歯周病の予防・治療研究に有用である。
A similar example is a report in which a three-dimensional oral mucosa model was infected with oral Candida fungus (Non-Patent Document 1).
However, there have been no reports on a method for inducing a periodontal disease-like state by infecting gingival tissue with periodontal pathogenic bacteria themselves.
The periodontal pathogenic bacteria in question generally prefer anaerobic conditions, and it is thought that they are difficult to grow under the culture conditions of the three-dimensional gingival model. In addition, unlike the oral mucosa model, the gingival epithelium model has a keratinized layer , i.e., keratinized tissue, so it was thought that it would be difficult for periodontal pathogenic bacteria to easily invade.
Therefore, we devised a method to create a model of periodontal disease-like conditions infected with periodontal pathogenic bacteria using a three-dimensional gingival epithelium model. If we can create a model that is closer to the periodontal disease-like conditions infected with periodontal pathogenic bacteria, it will be useful for research on the prevention and treatment of periodontal disease.
上述の通り、近年では、三次元歯肉上皮モデルを用いて歯周病原性細菌自体を感染させたモデルの作製は成し遂げられていない。三次元歯肉上皮モデルは角化した層、すなわち、角化組織を有しており、角化組織を通過して歯周病原性細菌を感染させるためには、菌の活性を高い状態に維持する必要があると考えられた。
代表的な歯周病原性細菌は、嫌気性菌に属するが、三次元歯肉上皮モデルは嫌気環境下で培養しないため、嫌気性を好む歯周病原性細菌の感染は困難であると考えられた。
本発明は、歯周病原性細菌を感染させた三次元歯肉上皮モデルの製造方法を提供することを目的とする。
As mentioned above, in recent years, the creation of a model infected with periodontal pathogenic bacteria itself using a three-dimensional gingival epithelium model has not been achieved. The three-dimensional gingival epithelium model has a keratinized layer , i.e., keratinized tissue , and it was thought that in order for periodontal pathogenic bacteria to infect through the keratinized tissue , it was necessary to maintain the activity of the bacteria at a high level.
Typical periodontal pathogenic bacteria belong to the anaerobic group, but because the three-dimensional gingival epithelium model is not cultured in an anaerobic environment, it was thought that infection by anaerobic periodontal pathogenic bacteria would be difficult.
An object of the present invention is to provide a method for producing a three-dimensional gingival epithelium model infected with periodontal pathogenic bacteria.
本発明者らは、嫌気性の歯周病原性細菌を感染させるため、嫌気性条件下にて、抗菌剤フリーの培地で培養を行うことにより、嫌気性の歯周病原性細菌が感染した歯周病様状態の三次元歯肉上皮モデルを構築できることを見出した。また、感染させる際に、病原因子を含有させることで、歯肉組織内への感染を増強できることを見出した。
すなわち、本発明の一形態によれば、三次元歯肉上皮モデルの組織内に嫌気性の歯周病原性細菌を進入させた歯周病様状態に誘導された三次元歯肉上皮モデルの製造方法が提供される。
The present inventors have found that a three-dimensional gingival epithelium model in a periodontal disease-like state infected with anaerobic periodontal pathogenic bacteria can be constructed by culturing the bacteria in an antibacterial agent-free medium under anaerobic conditions in order to infect the bacteria with anaerobic periodontal pathogenic bacteria. They have also found that the infection into the gingival tissue can be enhanced by adding a pathogenic factor during the infection.
That is, according to one aspect of the present invention, there is provided a method for producing a three-dimensional gingival epithelium model induced into a periodontal disease-like state by invading anaerobic periodontal pathogenic bacteria into the tissue of the three-dimensional gingival epithelium model.
本発明によれば、三次元歯肉上皮モデルにおいて嫌気性の歯周病原性細菌が感染した歯周病様状態を示す三次元歯肉上皮モデルの製造方法を提供することができる。 The present invention provides a method for producing a three-dimensional gingival epithelium model that exhibits a periodontal disease-like condition caused by infection with anaerobic periodontal pathogenic bacteria.
以下、本発明を実施するための形態を詳細に説明するが、本発明の技術的範囲は下記の形態のみに限定されることはない。 The following describes in detail the embodiments of the present invention, but the technical scope of the present invention is not limited to the embodiments described below.
＜歯周病原性細菌を感染させた歯周病様状態の三次元歯肉上皮モデルの製造方法＞
本発明の一形態は、正常状態の三次元歯肉上皮モデルの角化組織側もしくは培地側に、歯周病原性細菌を、又は歯周病原性細菌及び病原因子を添加し、嫌気性条件下にて培養する、三次元歯肉上皮モデルの組織内に歯周病原性細菌を感染させた歯周病様状態に誘導された三次元歯肉上皮モデルの製造方法である。
三次元歯肉上皮モデルとは、ヒト歯肉上皮にきわめて類似した組織学的形態を有する人工再生組織モデルであり、角化組織を有する。
<Method for producing a three-dimensional gingival epithelium model in a periodontal disease-like state infected with periodontal pathogenic bacteria>
One form of the present invention is a method for producing a three-dimensional gingival epithelial model induced to a periodontal disease-like state by infecting the tissue of the three-dimensional gingival epithelium model with periodontal pathogenic bacteria, or periodontal pathogenic bacteria and pathogenic factors, by adding the bacteria to the keratinized tissue side or culture medium side of a normal three-dimensional gingival epithelium model and culturing it under anaerobic conditions.
The three-dimensional gingival epithelium model is an artificial regenerated tissue model that has a histological morphology very similar to that of human gingival epithelium and contains keratinized tissue.
三次元歯肉上皮モデルは、歯肉上皮細胞をポリカーボネートフィルター上などで気液界面培養することにより構築されたものであることが好ましい。これにより、健常なヒト歯肉上皮にきわめて類似した組織学的形態を有する三次元歯肉上皮モデルを製造することができる。また、歯肉上皮モデルが三次元の組織モデルであること、すなわち歯肉上皮細胞が積層されていることは、顆粒層でのフィラグリンの発現、基底層上部でのケラチン６、ケラチン１０、ケラチン１３、ケラチン１６の発現などの組織学的な形態を確認することで判断することができる。このような正常状態の三次元歯肉上皮モデルの市販品としては、ＳｋｉｎＥｔｈｉｃ（登録商標） ＨＧＥ（株式会社ニコダームリサーチ）などが挙げられる。
本発明に係る製造方法では、上記正常状態の三次元歯肉上皮モデルを、歯周病原性細菌及び病原因子を角層側、すなわち、角化組織側から添加もしくはこれらを含む培地で、嫌気条件下にて培養することを有する。これにより、効果的に歯周病原性細菌を歯肉上皮細胞に感染させ、歯周病様状態へと誘導することができる。感染とは、組織内に侵入し組織が脆弱になる意味合いを含む。
The three-dimensional gingival epithelium model is preferably constructed by culturing gingival epithelial cells at an air-liquid interface on a polycarbonate filter or the like. This allows the production of a three-dimensional gingival epithelium model having a histological morphology very similar to that of healthy human gingival epithelium. In addition, the fact that the gingival epithelium model is a three-dimensional tissue model, i.e., that the gingival epithelium cells are layered, can be determined by confirming histological morphology such as the expression of filaggrin in the granular layer, and the expression of keratin 6, keratin 10, keratin 13, and keratin 16 in the upper part of the basal layer. Examples of such a commercially available three-dimensional gingival epithelium model in a normal state include SkinEthic (registered trademark) HGE (NicoDerm Research Co., Ltd.).
The manufacturing method according to the present invention comprises culturing the above-mentioned three-dimensional gingival epithelium model in a normal state under anaerobic conditions in a medium containing or to which periodontal pathogenic bacteria and pathogenic factors are added from the stratum corneum side , i.e., the keratinized tissue side . This makes it possible to effectively infect gingival epithelial cells with periodontal pathogenic bacteria and induce a periodontal disease-like state. The term "infection" includes the meaning of invading tissue and weakening the tissue.
歯周病原性細菌としては、歯周病に関連する嫌気性の細菌を使用できる。病原細菌としては、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ）、トレポネーマ・デンティコーラ（Ｔｒｅｐｏｎｅｍａ ｄｅｎｔｉｃｏｌａ）、タンネレラ・フォーサイシア（Ｔａｎｎｅｒｅｌｌａ ｆｏｒｓｙｔｈｉａ）、アグリゲイティバクター・アクチノミセテムコミタンス（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）、プレボテラ・インターメディア（Ｐｒｅｖｏｔｅｌｌａ ｉｎｔｅｒｍｅｄｉａ）などが挙げられる。好ましくは、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ）、トレポネーマ・デンティコーラ（Ｔｒｅｐｏｎｅｍａ ｄｅｎｔｉｃｏｌａ）、タンネレラ・フォーサイシア（Ｔａｎｎｅｒｅｌｌａ ｆｏｒｓｙｔｈｉａ）である。
培地に含まれる歯周病原性細菌の量は、歯周病様状態を誘導することができれば特に制限されないが、例えば１．０×１０４～１．０．×１０９ＣＦＵ／モデルであり、好ましくは１．０×１０６～１．０×１０8ＣＦＵ／モデルである。
The periodontal pathogenic bacteria may be anaerobic bacteria associated with periodontal disease. Examples of the pathogenic bacteria include Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Prevotella intermedia. Preferred are Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia.
The amount of periodontal pathogenic bacteria contained in the medium is not particularly limited as long as it can induce a periodontal disease-like condition, but is, for example, 1.0 x 10 4 to 1.0 x 10 9 CFU/model, and preferably 1.0 x 10 6 to 1.0 x 10 8 CFU/model.
病原因子としては、歯周病を誘導できる物質であれば、特に制限されず、例えばリポ多糖（ＬＰＳ）などの細菌内毒素、膜タンパク（ＯＭＰ）、Ｐｏｌｙｉｎｏｓｉｎｅ－ｐｏｌｙｃｙｔｉｄｙｌｉｃ ａｃｉｄ（Ｐｏｌｙ（Ｉ：Ｃ））などの病原細菌の構成成分などが挙げられる。これらの病原因子としては、細菌由来のものを使用できる。病原因子が由来する細菌としては、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ ｇｉｎｇｉｖａｌｉｓ）、トレポネーマ・デンティコーラ（Ｔｒｅｐｏｎｅｍａ ｄｅｎｔｉｃｏｌａ）、タンネレラ・ファーサイシア（Ｔａｎｎｅｒｅｌｌａ ｆｏｒｓｙｔｈｉａ）、アグリゲイティバクター・アクチノミセテムコミタンス（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）、プレボテラ・インターメディア（Ｐｒｅｖｏｔｅｌｌａ ｉｎｔｅｒｍｅｄｉａ）、フソバクテリウム・ヌクレアタム（Ｆｕｓｏｂａｃｔｅｒｉｕｍ ｎｕｃｌｅａｔｕｍ）などの歯周病原性細菌、及び大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）などが挙げられる。病原因子は、好ましくはＬＰＳ、ＯＭＰ及びＰｏｌｙ（Ｉ：Ｃ）から選択される少なくとも一つであり、より好ましくはＬＰＳである。
病原因子は、ヒト口腔内から採取された口腔内細菌やヒト体内から採取された病原細菌、これらの細菌由来の成分などを超音波処理により微細化する方法で調製することができる。また、市販品を用いることもでき、例えばＬｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ，ｆｒｏｍ Ｅ．ｃｏｌｉ Ｏ１１１（富士フイルム和光純薬株式会社）、ＬＰＳ－ＰＧ（ＬＰＳ Ｐ.ｇｉｎｇｉｖａｌｉｓ由来）（ナカライテスク株式会社）などが挙げられる。
培地に含まれる病原因子の量は、特に制限されないが、例えば０．０１～１０００μｇ／ｍＬであり、好ましくは１０～５００μｇ／ｍＬであり、より好ましくは５０～２００μｇ／ｍＬである。
The pathogenic factor is not particularly limited as long as it is a substance capable of inducing periodontal disease, and examples thereof include bacterial endotoxins such as lipopolysaccharide (LPS), membrane proteins (OMP), components of pathogenic bacteria such as polyinosine-polycytidylic acid (Poly(I:C)), etc. As these pathogenic factors, those derived from bacteria can be used. Examples of bacteria from which pathogenic factors are derived include periodontal pathogenic bacteria such as Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, and Fusobacterium nucleatum, as well as Escherichia coli. The virulence factor is preferably at least one selected from LPS, OMP and Poly(I:C), and more preferably LPS.
The pathogenic factor can be prepared by a method of micronizing oral bacteria collected from the human oral cavity, pathogenic bacteria collected from the human body, components derived from these bacteria, etc. by ultrasonic treatment. In addition, commercially available products can also be used, such as Lipopolysaccharide from E. coli O111 (FUJIFILM Wako Pure Chemical Industries, Ltd.) and LPS-PG (LPS derived from P. gingivalis) (Nacalai Tesque, Inc.).
The amount of the pathogenic factor contained in the medium is not particularly limited, but is, for example, 0.01 to 1000 μg/mL, preferably 10 to 500 μg/mL, and more preferably 50 to 200 μg/mL.
培地としては、三次元歯肉上皮モデルを維持培養できる維持培地であれば、特に制限されないが抗菌剤フリーの培地を使用する必要がある。例えば、三次元歯肉上皮モデルとしてＳｋｉｎＥｔｈｉｃ（登録商標） ＨＧＥ（株式会社ニコダームリサーチ）を使用する場合、同モデル専用である抗菌剤フリーのＭＡＩＮＴＥＮＡＮＣＥ ＭＥＤＩＵＭや抗菌剤フリーのＧＲＯＷＴＨ ＭＥＤＩＵＭを維持培地として使用できる。 As for the medium, there is no particular limitation as long as it is a maintenance medium that can maintain and culture the three-dimensional gingival epithelium model, but it is necessary to use an antibacterial agent-free medium. For example, when SkinEthic (registered trademark) HGE (NicoDerm Research, Inc.) is used as the three-dimensional gingival epithelium model, antibacterial agent-free MAINTENANCE MEDIUM or antibacterial agent-free GROWTH MEDIUM, which are exclusive to this model, can be used as the maintenance medium.
三次元歯肉上皮モデルの培養方法は、特に制限されず、培養する三次元歯肉上皮モデルに応じて適宜選択できる。ヒト歯肉上皮細胞を用いた三次元歯肉上皮モデルの場合、培養温度は、通常、３６～３８℃程度であり、好ましくは約３７℃である。培地のｐＨは、特に制限されないが、通常６～８程度である。培養時間は、特に制限されず、例えば歯周病原性細菌を接種して２時間～７日間嫌気条件にて維持して培養を行う。
嫌気性条件にする方法としては、酸素がない環境であれば、特に制限されないが、例えば嫌気性のグローブボックス、嫌気性チャンバー、密閉容器に脱酸素剤を入れる方法や、真空ポンプにて空気を吸引する方法などが挙げられる。
The method for culturing the three-dimensional gingival epithelium model is not particularly limited and can be appropriately selected depending on the three-dimensional gingival epithelium model to be cultured. In the case of a three-dimensional gingival epithelium model using human gingival epithelial cells, the culture temperature is usually about 36 to 38° C., and preferably about 37° C. The pH of the medium is not particularly limited, but is usually about 6 to 8. The culture time is not particularly limited, and for example, periodontal pathogenic bacteria are inoculated and cultured under anaerobic conditions for 2 hours to 7 days.
The method for creating anaerobic conditions is not particularly limited as long as it is an oxygen-free environment, and examples thereof include an anaerobic glove box, an anaerobic chamber, a method in which an oxygen scavenger is placed in a sealed container, and a method in which air is sucked in using a vacuum pump.
正常状態の三次元歯肉上皮モデルが歯周病様状態に誘導されたことは、歯肉上皮細胞における歯周病原性細菌が歯肉上皮組織の角化層を通過して組織内部に感染することにより確認できる。
したがって、本発明に係る製造方法においては、上記誘導工程後、歯周病原性細菌の感染を免疫染色法などにより観察する工程を有する。さらに、歯肉細胞の組織学的な変化を測定または観察する工程を含む場合もある。
The induction of a normal three-dimensional gingival epithelial model into a periodontal disease-like state can be confirmed by observing that periodontal pathogenic bacteria in gingival epithelial cells pass through the keratinized layer of the gingival epithelial tissue and infect the inside of the tissue.
Therefore, the production method according to the present invention includes a step of observing the infection by periodontal pathogenic bacteria by immunostaining after the induction step, and may further include a step of measuring or observing histological changes in gingival cells.
本発明の一実施形態は、本発明に係る製造方法により製造された、歯周病様状態の三次元上皮モデルである。歯周病様状態の三次元上皮モデルは、ｉｎ ｖｉｔｒｏでの歯周病様状態を再現することができ、ヒトや動物を用いることなく、個体値や歯周病の程度のバラツキが抑えられ、再現性の高い試験評価を行うことができる。また、安定的に複数のモデルに対して歯周病様状態を再現できることから、歯周病に対する予防・治療に関わる成分および／または改善作用のある成分を含む薬剤や組成物がもつ作用の評価や開発を効率よく行うことができる。 One embodiment of the present invention is a three-dimensional epithelial model of a periodontal disease-like state produced by the production method of the present invention. The three-dimensional epithelial model of a periodontal disease-like state can reproduce a periodontal disease-like state in vitro, and can perform highly reproducible test evaluations with reduced variation in individual values and the degree of periodontal disease without using humans or animals. In addition, because the periodontal disease-like state can be stably reproduced in multiple models, it is possible to efficiently evaluate and develop the effects of drugs and compositions containing components related to the prevention and treatment of periodontal disease and/or components with improving effects.
以下に実施例を挙げて本発明を具体的に説明するが、本発明の技術的範囲がこれに限定されるものではない。 The present invention will be specifically explained below with reference to examples, but the technical scope of the present invention is not limited to these examples.
実施例１：三次元歯肉上皮モデルを歯周病原性細菌および病原因子を含む培地で嫌気性条件下にて培養し、組織内に歯周病原性細菌を感染させた歯周病様状態の三次元歯肉上皮モデルの作製
１．試験概要
三次元歯肉上皮モデルをＰ．ｇｉｎｇｉｖａｌｉｓおよびＬＰＳを含む培地で嫌気性条件下にて培養し、組織内にＰ．ｇｉｎｇｉｖａｌｉｓを感染させた三次元歯肉上皮モデルを作製し、組織学上の変化を確認した。
Example 1: Preparation of a three-dimensional gingival epithelial model in a periodontal disease-like state by culturing a three-dimensional gingival epithelial model under anaerobic conditions in a medium containing periodontal pathogenic bacteria and pathogenic factors and infecting the tissue with periodontal pathogenic bacteria 1. Test overview A three-dimensional gingival epithelial model was cultured under anaerobic conditions in a medium containing P. gingivalis and LPS to prepare a three-dimensional gingival epithelial model in which the tissue was infected with P. gingivalis, and histological changes were confirmed.
２．試験試料
三次元歯肉上皮モデル：ＳｋｉｎＥｔｈｉｃ（登録商標） ＨＧＥ（株式会社ニコダームリサーチ）
歯周病原性細菌：Ｐ．ｇｉｎｇｉｖａｌｉｓ ＪＣＭ１２２５７
細菌内毒素：Ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ， ｆｒｏｍ Ｅ．ｃｏｌｉ Ｏ１１１（富士フイルム和光純薬株式会社）
ＰＢＳ（－）：リン酸緩衝生理食塩水。
2. Test sample Three-dimensional gingival epithelium model: SkinEthic (registered trademark) HGE (NicoDerm Research Co., Ltd.)
Periodontal pathogenic bacteria: P. gingivalis JCM12257
Bacterial endotoxins: Lipopolysaccharide, from E. coli O111 (FUJIFILM Wako Pure Chemical Industries, Ltd.)
PBS(-): phosphate buffered saline.
３．試験方法
ＳｋｉｎＥｔｈｉｃ（登録商標） ＨＧＥ（以下、「ＨＧＥ」とも称す）を製造会社のプロトコルに従って、維持培地（ＭＡＩＮＴＥＮＡＮＣＥ ＭＥＤＩＵＭ）を添加したプレートにて馴化した。培地として、維持培地（コントロール）もしくは、２００μｇ/ｍＬのＬｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ， ｆｒｏｍ Ｅ．ｃｏｌｉ Ｏ１１１（以下、「Ｅ．ｃｏｌｉ ＬＰＳ」とも称す）を含有する維持培地を別のプレートに２ｍＬ添加して、馴化したＨＧＥを設置した。設置後、１５０μＬのＰ．ｇｉｎｇｉｖａｌｉｓ懸濁液をＨＧＥ上部に添加して、３７?Ｃの嫌気性条件下にて培養を開始した。嫌気性条件は、培養プレートを嫌気チャンバーに入れたのち、脱酸素剤を封入して設定した。
６時間培養後、ＰＢＳ（－）でＨＧＥの上部を洗浄し、Ｐ．ｇｉｎｇｉｖａｌｉｓを除去した後、新しい維持培地を１５０μＬ添加し、３７?Ｃの嫌気性条件下で培養した。
４８時間培養後、ＨＧＥから凍結切片を作成してＰ．ｇｉｎｇｉｖａｌｉｓに対する抗体を用いて免疫染色し、組織切片像を顕微鏡（カールツァイス社製、倍率：１００）で観察した。
3. Test Method SkinEthic® HGE (hereinafter also referred to as "HGE") was acclimatized on a plate containing maintenance medium (MAINTENANCE MEDIUM) according to the manufacturer's protocol. 2 mL of maintenance medium (control) or maintenance medium containing 200 μg/mL Lipopolysaccharide from E. coli O111 (hereinafter also referred to as "E. coli LPS") was added to another plate as a medium, and the acclimatized HGE was installed. After installation, 150 μL of P. gingivalis suspension was added to the top of the HGE, and culture was started under anaerobic conditions at 37°C. Anaerobic conditions were set by placing the culture plate in an anaerobic chamber and then sealing an oxygen scavenger.
After 6 hours of culture, the top of the HGE was washed with PBS(-) to remove P. gingivalis, and then 150 μL of fresh maintenance medium was added and cultured under anaerobic conditions at 37°C.
After 48 hours of culture, frozen sections were prepared from the HGE and immunostained with an antibody against P. gingivalis, and the images of the tissue sections were observed under a microscope (Carl Zeiss, magnification: 100).
４．結果
免疫染色後の組織切片像を図１に示す。好気性条件下で、LPSを含む培地で培養した場合Ｐ．ｇｉｎｇｉｖａｌｉｓは組織の上部で検出され組織内部ではほとんど検出されなかった。一方、嫌気性条件下で培養した場合では、Ｐ．ｇｉｎｇｉｖａｌｉｓの組織内への侵入が観察された。さらに、嫌気性条件下でかつ、LPSを含む培地で培養した場合には、Ｐ．ｇｉｎｇｉｖａｌｉｓの組織内部への侵入が観察された。組織切片像においても、組織の厚さが薄く脆弱に変化している様子も確認された（図１）。
Ｐ．ｇｉｎｇｉｖａｌｉｓの接種6時間に比較して、Ｐ．ｇｉｎｇｉｖａｌｉｓ除去後の48時間培養後においてＰ．ｇｉｎｇｉｖａｌｉｓのＨＧＥ組織内での増殖が観察された。このことから、組織内に感染したＰ．ｇｉｎｇｉｖａｌｉｓが組織内で増殖していることが確認される（図１）。
４８時間培養後のＨＧＥの細胞生存率をアラマブルー法にて評価したところ、細胞生存率への影響は認められなかった。
4. Results Images of tissue sections after immunostaining are shown in Figure 1. When cultured in a medium containing LPS under aerobic conditions, P. gingivalis was detected in the upper part of the tissue and was hardly detected inside the tissue. On the other hand, when cultured under anaerobic conditions, invasion of P. gingivalis into the tissue was observed. Furthermore, when cultured in a medium containing LPS under anaerobic conditions, invasion of P. gingivalis into the tissue was observed. Images of tissue sections also confirmed that the tissue had become thinner and more fragile (Figure 1).
Compared to 6 hours after inoculation of P. gingivalis, P. gingivalis was observed to grow in the HGE tissue 48 hours after removal of P. gingivalis, confirming that P. gingivalis infected the tissue and grew therein (Fig. 1).
When the cell viability of HGE after 48 hours of culture was evaluated by the Alama Blue method, no effect on the cell viability was observed.
本発明は、三次元ヒト歯肉上皮モデルにおいて歯周病原性細菌を感染させる手段を提供することができる。 The present invention can provide a means for infecting a three-dimensional human gingival epithelium model with periodontal pathogenic bacteria.
１ 三次元歯肉上皮モデル（角化組織側もしくは培地側に歯周病原性細菌を接種）
２ 三次元歯肉上皮モデルのウェル
３ 培地ウェル（プレート）
４ 培地
５ 脱酸素剤を封入した嫌気チャンバー
６ 脱酸素剤
1. Three-dimensional gingival epithelium model (periodontal pathogenic bacteria inoculated on the keratinized tissue side or the culture medium side)
2. Well of three-dimensional gingival epithelium model 3. Culture medium well (plate)
4 Culture medium 5 Anaerobic chamber containing oxygen absorber 6 Oxygen absorber
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